Validated HPLC Method with UV Detection for Florfen- icol Determination in Bester Sturgeon, A Cultured Hy- brid of Huso Huso × Acipenser ruthenus

The hybrid Bester sturgeon (Huso huso × Acipenser ruthenus) is one of the most important commercial fish species in the world. Farming of sturgeon in worldwide has increased in recent years and becomes an important issue in the wider context of global food supplies [1]. In Taiwan, the techniques for sturgeon culturing have been established in several fish farms. However, the high density of animals grown in agricultural facilities and fish hatcheries in either largeor small-scale fish farms increases the potential for disease outbreak. To minimize the impact of an outbreak spreading across an animal population, the use of drugs, especially antibiotics, in sturgeon aquaculture becomes very important [2]. Florfenicol (FFC), a fluorinated analogue of thiamphenicol and chloramphenicol, has Received: April 18, 2019


Introduction
The hybrid Bester sturgeon (Huso huso × Acipenser ruthenus) is one of the most important commercial fish species in the world.
Farming of sturgeon in worldwide has increased in recent years and becomes an important issue in the wider context of global food supplies [1]. In Taiwan, the techniques for sturgeon culturing have been established in several fish farms. However, the high density of animals grown in agricultural facilities and fish hatcheries in either large-or small-scale fish farms increases the potential for disease outbreak. To minimize the impact of an outbreak spreading across an animal population, the use of drugs, especially antibiotics, in sturgeon aquaculture becomes very important [2]. Florfenicol (FFC), a fluorinated analogue of thiamphenicol and chloramphenicol, has a synthetically produced broad antibacterial spectrum similar to chloramphenicol and stronger than thiamphenicol [3]. It has been widely used in veterinary as well as in aquaculture.
Among 14 antibiotics authorized for application in Taiwan aquaculture, FFC is only available for sturgeon aquaculture.
Nevertheless, there is a lack of understanding regarding FFC metabolism kinetics during breeding period. The overall goal is to determine the presence and distribution of FFC residue in Bester sturgeon after treatment to obtain information about FFC withdrawal period for sturgeon culturing. This paper and video describe the stepwise development of a simple, rapid, and reliable method for detecting the pharmacokinetics of FFC in serum, muscle and liver of Bester sturgeon. The details of sampling and assay validation methods as well as the representative results are shown to demonstrate the application of high-performance liquid chromatography (HPLC) method with ultraviolet (UV) detector.
The simple and reliable technique can be utilized to design optimal dosage regiments for aquatic veterinary applications.    d. Approximately 0.5 mL of blood samples were obtained from the caudal artery of each fish (Figure 3).

Materials and Methods
e. Blood samples were allowed to clot at room temperature for 30 min then the coagulated blood samples were centrifuged at 1,300 ×g for 10 min.
f. Sera were collected and stored at -80˚C.
g. The muscle and liver samples were excised and snapfrozen at -80˚C ( Figure 4).
h. Frozen muscle and liver samples were homogenized in buffer.
i. The tissue slurry was then centrifuged at 5,000 rpm for 30 min at 4°C, the supernatants were collected and kept at -80˚C for subsequent analysis.

Method Validation [4]
a. Linearity was assayed according to the slopes, intercepts.
Ten mg mL -1 FFC stock solution in methanol was diluted into working solutions of 100, 10, and 1 μg mL -1 with distilled water.
b. Standardization was performed by a high-concentration and a low-concentration range.
c. Blank sera, muscle and liver extract samples were exposed to known amounts of FFC (0.1, 0.2, 0.5, 1, 2, 5, and 10 μg mL -1 ) and were transferred to autosampler tubes for direct application to the HPLC column. d.
The presence of FFC in serum and tissue samples was screened by HPLC method with ultraviolet photodiode array detection. e.
The internal standard was analyzed as the unknown samples.
f. The FFC concentrations in unknown samples were read from the standard curve.
g. Accuracy was evaluated indirectly because of the lack of certified samples or reference methods. h.
Comparing with the resulting peak area ratios of blank sera, muscle and liver extract samples with known amounts of FFC (0.1, 0.2, 0.5, 1, 2, 5, and 10 μg mL -1 ) and water samples containing equivalent concentrations of FFC, the difference between these two sets of data was the bias of the method.
i. The limit of detection (LOD) and limit of quantification (LOQ) for FFC were estimated from the size of the FFC peak in spiked samples of blank sera, muscle and liver extract samples.
They were defined as the concentrations that resulted in a detectable peak of approximately 4 and 10 times of the noise level, respectively.

Statistical Analysis
Data are shown as mean ± the standard error of the mean (SEM). Student's t-test was used for statistical analyses. Differences between groups were considered statistically significant at *p < 0.05.  b. Figure 6. Serum FFC concentrations after single oral FFC administration. c. Figure 7. FFC concentration time curve in liver and muscle following single oral FFC administration at doses of (A) 10 mg kg -1 BW and (B) 20 mg kg -1 BW.

For the Results Presentation-There is One Session 2:40 min Long
d.    c. According to Taiwan law in the allowed amount of FFC residue in the fish meat must be ≤ 1 µg mL-1. d.
The recommended withdrawal period of FFC in sturgeon was suggested for 15 days.

Representative Results
As shown in Table 1 (Table 1).

Discussion
The present work demonstrated a method for determining FFC retention and elimination in sturgeon culturing.  [9]. From a therapeutic point of view, the higher oral bioavailability and slower elimination of FFC than that in most antibiotics (amoxicillin, erythromycin, flumequine, lincomycin, oxalinic acid, oxytetracycline, and spiramycin) are important properties for treating with bacterial infections in aquaculture. However, antibiotic residues in animal-derived food stuffs cause multi-drug resistance of pathogens for antibiotics used in human medicine and bear a risk for the food-productionindustry [10].
As a result, the concerns over veterinary drug residues in food are increased globally. Various methods have been described for identifying FFC in animal tissues, including HPLC [11], liquid chromatography-mass spectrometry [12,13], gas chromatography [14], gas chromatography-mass spectrometry [15], and enzymelinked immunosorbent assay [16]. The HPLC and LC-MS/MS methods are widely analytical tools in pharmacokinetic study in several fish species. FFC residues in fish muscle have been monitored by HPLC-UV method, and results were close with those obtained using liquid chromatography-tandem quadrupole mass spectrometry. HPLC-UV method can be employed for the analysis of FFC metabolic kinetics simply and reliably regardless of different species and environmental condition [17]. The total concentration of residues of FFC and FFC amine in fish accepted by European Union were not exceeding 1,000 µg kg -1 [18]. In Taiwan, maximum residue limits for veterinary drugs allowed in fish is 1 μg mL -1 .
Our results showed that FFC concentrations in muscle and liver decreased slowly with time and were below the limit of detection at 10 and 14 days after oral administration, respectively. Accordingly, withdrawal periods of 15 days after oral administration of FFC in sturgeon are recommended for fish farm. These findings can provide valuable insight for designing optimal dosage regimens for FFC in aquatic agricultural system.